Biological material is often kept at low temperatures to prevent damage that may be caused from biological processes during storage. For a relatively short period of storage (normally up several to weeks) the material may be kept at low temperatures that are above freezing (hypothermic preservation). For example, red blood cells (RBC or erythrocytes) are usually stored for up to 42 days in a refrigerator at about 4° C., after which they must be discarded because RBC recovery falls below acceptable levels.
Preservation at a temperature below 0° C. (defined herein as “cryopreservation”), allows much longer storage times and may be at any temperature below 0° C., including such temperatures below −20° C., −70° C., −135° C., or in liquid nitrogen. Cryopreservation is achievable by freezing or by vitrification. In vitrification, ice-crystals are not formed, however high concentrations of potentially toxic cryoprotectant agents must be added to the biological material. These cryoprotectant agents must be removed before the biological sample is used, in order not to harm the recipient of the biological material. Freezing is also known to cause damage. For example, ice crystals forming in the solution exert extra-cellular mechanical stress. Intracellular stress can be caused for example by osmosis of water into the extra-cellular space, to replace water that is already frozen.
One factor that has a major effect on the success of cryopreservation is the composition of the solution in which the biological material is immersed prior to freezing. Currently many different cryopreservation solutions are known. Normally, such solutions contain a balanced salt solution such as phosphate buffered saline (PBS) and cryoprotectant agents (CPAs). Most freezing solutions comprise cryoprotectants selected from dimethyl sulfoxide (DMSO) and one or more polyalcohols such as glycerol, ethylene glycol, propylene glycol, and other molecules including butandiol and methanol. In addition, sugars, proteins, carbohydrates such as hydroxy ethyl starch (HES), dextran and other macromolecules are also used. Trehalose, for example, is thought to be protective by binding to lipid polar groups and replacing water. In addition, in WO 99/60849 for example it was claimed that addition of biochemistry altering reagents would reduce hemolysis of RBC during the freeze-thaw cycle.
For example, the current freezing method for RBC employs the addition of glycerol at high concentrations. There are two main methods for freezing with glycerol: high glycerol method (HGM) where the final concentration of glycerol is almost 40% and low glycerol method (LGM) where the final glycerol concentration is 19%. When HGM is used storage is done at −80° C. freezers, whereas, when using LGM storage is done in LN (−196° C.). Since, glycerol is toxic it has to be washed out after thawing. This is done in a process known as “de-glycerolization”, wherein the thawed RBC are centrifuged, the supernatant removed and the pellet suspended in a glycerol free solution. This is normally repeated 3 times with decreasing sodium chloride concentrations (staring with 12% NaCl, then 1.6% NaCl and 0.9% NaCl) in order that intra-cellular glycerol be removed. Furthermore, as this process typically takes about 60 minutes, it renders frozen blood supplies an impractical solution for emergency use.
One problem associated with preservation of RBC is free hemoglobin in the sample, which increases with storage time and with damage to RBC. Free hemoglobin is hemoglobin that is not within an RBC and is considered undesirable in RBC samples for transfusion, not only because it is indicative of hemolysis (i.e. damage to RBC) but also since it in itself is hazardous to the recipient. It is normally calculated as the percent of supernatant hemoglobin from the total amount of hemoglobin in the sample. Current regulatory requirements in the United States from RBC for transfusion include that there be less then 1% free hemoglobin in the serum and that 75% of the cells need to be circulating 24 h after transfusion in vivo (De Korte et al. 2004). European regulations place a higher demand—that free hemoglobin be less than 0.8%.
The post-thaw hemolysis measured after thawing (but before washing) of RBC frozen in HGM or LGM are 4.4±1.0% and 10±2.0%, respectively (Lawns et al. 2003). Only after de-glycerolization (i.e. washing of the thawed cells) do the thawed RBC answer the quality demands for blood to be transfused. In WO 99/60849, frozen blood has less free hemoglobin, however this result is probably attributable to the use of (a) glycerol, (b) DMSO or (c) simultaneous use of many different CPAs.
In most cryopreservation protocols, preservation of the frozen biological material is at a temperature below −130° C. This is normally done in containers of liquid nitrogen (LN) by either immersion of the biological material in LN or in LN vapor. This adds significantly to the cost of long-term preservation. In addition, incidents are known where the LN in the container evaporated (either due to a malfunction of the container or human error) and the biological materials were damaged.
One method that can overcome these obstacles is lyophilization of the frozen biological material (e.g. WO 03/020874). Lyophilization is a process in which ice crystals are removed by sublimation and desorption, resulting in dry matter. The lyophilized material may be stored at room temperature for a long period of time and be rehydrated for use by simply adding water. Lyophilization results in higher survival rates than air drying or heating, but is still a damaging process.
In order to enhance the biological material's ability to survive the freeze-drying process, intercellular and/or extra-cellular lyoprotectant agents (LPAs) are often added to the biological material. One such LPA is trehalose. It was shown, for example, that loading of platelets with trehalose, or with trehalose and DMSO, may improve their ability to withstand cryopreservation without premature inactivation (e.g. U.S. Pat. No. 5,827,741 and U.S. Pat. No. 6,723,497).
Commercial lyophilization of RBC is not performed and there is no known method or product that has been approved for use today. Goodrich R. P et al. (1992) carried out lyophilization of RBC and evaluated their enzymatic activity. They found that after lyophilization, most RBC enzymes have essentially maintained their concentrations. However, the two major organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3-DPG), had reduced intra-cellular concentrations. In U.S. Pat. No. 4,874,690 free hemoglobin was not reported. However, the hemoglobin recovery level and the cell recovery levels were no more than 70%. This indicates that at least 30% of the cells fractured in these experiments and that the free hemoglobin was at least 30%.
Another approach to lyophilization of cells is the introduction of trehalose into cells. Trehalose is known to protect cell membranes in a dry state (Chen et al., 2001). It was also shown to improve platelet survival after freeze-drying (Crowe et al., 2003). Satpathy G R. et al. (2004), have introduced trehalose into RBC, although they did not attempt to freeze them, and evaluated the effect and loading method on RBC. They found that uptake of trehalose increases with its concentration, but that concomitantly hemolysis also increases significantly.
U.S. Pat. No. 6,770,478 discloses a method for the introduction of trehalose into cells by depletion of cholesterol from the RBC membrane, thus allowing for the trehalose to enter the cells. In this patent only membrane properties of RBC were evaluated and not the recovery of the cells (in terms of cells number and plasma free hemoglobin) after freeze-drying. However, Satpathy G. R. et al. (2004), have showed that introduction of trehalose into RBC causes significant hemolysis. In fact, this work showed that hemolysis was a result if introducing trehalose into the cells since hemolysis occurred in fresh RBC, since they did not freeze or freeze-dry the cells.
Apart from RBC, other cell types are known to populate blood. These are normally referred to as white blood cells (WBC), including monocytes, lymphocytes, granulocytes, platelets and macrophages. Lymphocytes (B lymphocytes and T lymphocytes), under normal conditions, make up about 20 to 35% of all white cells, but proliferate rapidly in the face of infection. Monocytes ordinarily number 4% to 8% of the white cells.
Umbilical cord blood (UCB) is a source for hematopoietic stem cells (HSC). HSC are cells that can differentiate into all blood cells. Other sources for HSC are bone marrow and very small amount of HSC can be found circulating in peripheral blood (as WBC). Morphologically, HSC have a round nucleus similar to the mononuclear white blood cells (lymphocytes and monocytes). They resemble lymphocytes very much, and may be slightly bigger. The method to differentiate between them is according to cell membrane antigens. HSC are normally identified by expression of the CD34 antigen. HSC (from peripheral blood, bone marrow or UCB) are given to patients whose immune systems has been damaged, e.g. due to chemotherapy and/or radiotherapyin and in different diseases such as: acute and chronic leukemias, myelodysplastic syndromes, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma, aplastic anemia, thalassemia, sickle cell anemia, neuroblastoma and more.
The current method for the preservation of HSC is using 10% DMSO and storage in liquid nitrogen (LN). When storing HSC from UCB and from peripheral blood the cells are separated using ficol-paque and the fraction that is stored are the MNC.
Epigallocatechin gallate (EGCG) is a polyphenol (MW 458.4) found naturally for example in green and black tea. The well-known beneficial effects associated with such tea are attributed, at least in part, to EGCG. Among the mechanisms associated with EGCG's beneficial effects are its ability to function as an antioxidant, its ability to associate with the phospholipids bi-layer of the cell membrane (Fujiki et al. 1999) and the lipid head groups of liposomes (Kumazawa et al., 2004) and more. Whilst EGCG is the main constituent of green tea, other polyphenols that are found naturally in green tea, such as epicatechin gallate (ECG) epigallocatechin (EGC) and epicatechin (EC), are also found in green tea and, like EGCG, are considered to be non-toxic. These polyphenols share structural and functional properties with EGCG (Suganuma et al. 1999).